Molecular tools have been extensively used in recent decades to examine biological
invasion processes, and are increasingly being adopted as efficient tools to support
non-indigenous species surveys, notably through barcoding approaches, i.e., the use
of a reference sequence specific to a given species to validate its identification. The
technique is easy to use but requires reliable reference sequences to be available in
public databases. In addition, the increasing discovery of cryptic species in marine taxa
may complicate taxonomic assignment. We illustrate these two issues in the ascidian
genus Botrylloides, in which at least three global marine invaders have been recognized,
including B. violaceus and B. diegensis. We obtained COI sequences from >750
colonies of Botrylloides spp. sampled in W Europe or provided by expert colleagues
from other regions. Phylogenetic trees clearly distinguished our targeted taxa [i.e.,
B. violaceus, B. diegensis and B. leachii (native)]. They also revealed another discrete
lineage apparently related to a recently described eastern Mediterranean species. By
examining public databases, we found sequences of B. diegensis erroneously assigned
to B. leachii. This observation has major implications as the introduced B. diegensis can
be misidentified as a putatively native species. We also checked published sequences
of the genus Botrylloides in the Mediterranean Sea, complemented with new samples.
Based on our custom reference database, all published sequences of B. leachii
corresponded to B. diegensis, although this NIS has hardly been reported at all in the
Mediterranean region. Such database errors are unfortunate, as the barcoding approach
is a powerful tool to identify the recognized Botrylloides species currently present in
European seas. This is of particular importance because a trait often used during
field assessment, i.e., single-color vs. two-color colonies, is misleading to distinguish
B. violaceus and B. diegensis respectively: a substantial proportion of the single-color
morph are actually B. diegensis in both the Mediterranean Sea and the English Channel.
Altogether, this study exemplifies the advantages and disadvantages of molecular
barcoding in NIS surveys and studies. The limitations that were identified are all easy
to resolve once proper vouchers and collections are set up.